P1425: ACTIVATED AND EXPANDED NATURAL KILLER CELLS TRANSDUCED WITH AN NKG2D CHIMERIC ANTIGEN RECEPTOR AGAINST ACUTE MYELOID LEUKEMIA
Authors: L. Córdoba, E. Castellano, A. Valeri, P. Rio, A. García-Ortiz, J. Encinas, E. Maroto, L. Fernández-Casanova, A. Pérez Martínez, D. Lee, D. J. Powell, A. Leivas, J. Martínez-López
Journal: HemaSphere (2022)

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy incurable for almost all patients. Chimeric Antigen Receptor (CAR) therapy is showing promising results in other hematological disorders but remains challenging in AML since no specific antigens have been described yet. Using NKG2D as a CAR, a natural NK receptor with 8 ligands overexpressed in several tumors, could surmount AML targeting limitations. T cells are considered the gold standard immune effector cells for CAR therapy, but they show toxicities that could be overcome using other cells such as activated and expanded natural killer cells (NKAE), that can be used in an allogeneic context with no GvHD, have natural anti-tumor properties and a short lifespan within the organism. In this project we analyze the in vitro efficacy and security of peripheral blood NKAE cells lentivirally transduced with an NKG2D-41BB-CD3z CAR. Aims: The primary objective of this project is to assess the efficacy and security of a cell immunotherapy against AML based on the use of NKAE cells transduced with an NKG2D CAR. Methods: NKAE cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (HD) and/or AML patients after 7 days of coculture with the irradiated cell line k562-mb21-41BBL by magnetic immunodepletion, when they were transduced with lentiviral vectors carrying NKG2D-41BB-CD3z CAR construct. CAR expression was measured up to 15 days post-transduction by flow cytometry. Cytotoxic activity against AML cell lines was performed by Propidium Iodide and Annexin V staining. Toxicity was evaluated by Europium-TDA assays. Results: AML can be targeted with an NKG2D CAR since all patients studied expressed at least one NKG2DL (Fig.1). Primary NK cells can be lentivirally transduced with an NKG2D CAR, showing a modest but stable CAR expression up to 15 days after transduction (23% ±4,7% NKG2D+, 33,35% ±4,25% GFP+). CAR-NKAE cells perform robust cytotoxicity towards MOLM-13 AML cell line after 24h of coculture at an effector:target ratio of 1:1, exerting a quasi-total lysis of AML cells (Fig.2A). This represents a significant increase in anti-leukemia effect compared to untransduced NKAE (92,6% ±0,3% vs 72,2% ±10%, p= 0,0138). Surface expression of relevant molecules for NK activity showed that NKp30, NKp44 (natural cytotoxicity receptors), CD69 (early activation marker), CD25 (IL2R alpha chain), FasL (mediates FasL-mediated cytotoxicity), NKp80 (C-type lectin-like surface-activating receptor) and TRAIL (TNF-Related Apoptosis Inducing Ligand) were more expressed by transduced NKAE (Fig.2B). Cytokine release profile revealed a higher production of IL-6, IL-17A, sFasL and IFNg by CAR-NKAE (Fig.2C). We did not observe relevant toxic effects of none of the cells against healthy PBMCs and very low toxicities were found against NL-20 lung cell line, but there were no differences between NKAE and CAR-NKAE. Image:Summary/Conclusion: Our preliminary results show that primary NK cells can be lentivirally transduced with a second generation NKG2D CAR, exerting a robust anti-leukemia activity towards AML cell lines. Transduced cells show a more cytotoxic immunophenotype as well as a higher release of cytolytic molecules and proinflammatory and proapoptotic proteins, that could be responsible of the increased anti-tumor response. Hence, NKG2D-CAR transduced NKAE cells could be a suitable approach to treat AML. Other costimulatory domains are being studied in order to assess which one triggers a higher cytotoxic activity, thus allowing decreasing the effector:target ratios and therefore, on-target off-tumor effect.